Antibodies with Potent and Broad Neutralizing Activity Against Antigenically Diverse and Highly Transmissible SARS-Cov-2 Variant

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bioRxiv preprint doi: https://doi.org/10.1101/2021.02.25.432969; this version posted March 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 andDraft is also Manuscript made available: forWang use under et al. a CC02021 license.

Antibodies with potent and broad neutralizing activity against antigenically diverse and

highly transmissible SARS-CoV-2 variants

Lingshu Wang1,†, Tongqing Zhou1,†, Yi Zhang1, Eun Sung Yang1, Chaim A. Schramm1, Wei

Shi1, Amarendra Pegu1, Olamide K. Oloninyi1, Amy Ransier1, Samuel Darko1, Sandeep R.

5 Narpala1, Christian Hatcher1, David R. Martinez2,3, Yaroslav Tsybovsky4, Emily Phung1,

Olubukola M. Abiona1, Evan M. Cale1, Lauren A. Chang1, Kizzmekia S. Corbett1, Anthony T.

DiPiazza1, Ingelise J. Gordon1, Kwanyee Leung1, Tracy Liu1, Rosemarie D. Mason1, Alexandra

Nazzari1, Laura Novik1, Adam S. Olia1, Nicole A. Doria-Rose1, Tyler Stephens4, Christopher D.

Stringham1, Chloe Adrienna Talana1, I-Ting Teng1, Danielle Wagner1, Alicia T. Widge1,

1 1 1 1 10 Baoshan Zhang , Mario Roederer , Julie E. Ledgerwood , Tracy J. Ruckwardt , Martin R.

Gaudinski1, Ralph S. Baric2,3, Barney S. Graham1, Adrian B. McDermott1, Daniel C. Douek1,

Peter D. Kwong1, John R Mascola1, Nancy J. Sullivan1,*, John Misasi1,†

Affiliations:

15 1Vaccine Research Center, National Institute of Allergy and Infectious Diseases, National

Institutes of Health, Bethesda, MD 20892, USA.

2Department of Epidemiology, UNC Chapel Hill School of Public Health, University of North

Carolina School of Medicine, Chapel Hill, NC 27599, USA.

3Department of Microbiology and Immunology, University of North Carolina School of

20 Medicine, Chapel Hill, NC 27599, USA.

4Electron Microscopy Laboratory, Cancer Research Technology Program, Leidos Biomedical

Research, Inc., Frederick National Laboratory for Cancer Research, Frederick, MD 21702, USA.

†Equal contributions

*Corresponding author: [email protected] 1

Abstract:

The emergence of highly transmissible SARS-CoV-2 variants of concern (VOC) that are resistant

to therapeutic antibodies highlights the need for continuing discovery of broadly reactive

5 antibodies. We identify four receptor-binding domain targeting antibodies from three early-

outbreak convalescent donors with potent neutralizing activity against 12 variants including the

B.1.1.7 and B.1.351 VOCs. Two of them are ultrapotent, with sub-nanomolar neutralization titers

(IC50 <0.0006 to 0.0102 µg/mL; IC80 < 0.0006 to 0.0251 µg/mL). We define the structural and

functional determinants of binding for all four VOC-targeting antibodies, and show that

10 combinations of two antibodies decrease the in vitro generation of escape mutants, suggesting

potential means to mitigate resistance development. These results define the basis of therapeutic

cocktails against VOCs and suggest that targeted boosting of existing immunity may increase

vaccine breadth against VOCs.

15 One Sentence Summary: Ultrapotent antibodies from convalescent donors neutralize and

mitigate resistance of SARS-CoV-2 variants of concern.

Main Text:

Since the start of the SARS-CoV-2 outbreak, >100 million people have been infected and

20 >2 million have died from COVID-19 (1). Shortly after the first Wuhan Hu-1 (WA-1) genome

sequence was published (2), spike proteins were generated for use in spike-specific antibody

discovery (3–5). Recently, virus variants first detected in the UK (e.g., B.1.1.7)(6), South Africa

(e.g., B.1.351) (7) and Brazil (P.1) (8, 9) have been shown to contain mutations that mediate

resistance to therapeutic monoclonal antibodies, have increased transmissibility and to potentially

increase pathogenicity (10–14). Additionally, vaccines designed based on the original WA-1

outbreak strain sequence elicit antibody responses that show decreased in vitro neutralizing

activity against variants (14–16). In this study, we investigated antibodies isolated from

5 convalescent subjects who were infected by the WA-1 strain during the first few months of the

outbreak, determined their reactivity against variants of concern (VOCs) and defined the structural

features of their binding to spike.

We obtained blood from four mild to moderately ill WA-1-infected subjects between 30

and 50 days after symptom onset. CD19+/CD20+/IgM-/IgA+ or IgG+ B cells were sorted for

10 binding to S-2P, receptor binding domain-subdomain-1 (RBD-SD1) or the S1 domain and

individual B-cell receptors were sequenced (Figure 1A, Figure S1). In total, we sorted 889 B cells

and recovered 709 (80%) paired heavy and light chain sequences and selected 200 antibodies for

expression. Among the 200 antibodies, there was a broad response across all spike domains with

77 binding RBD, 46 binding N-terminal domain (NTD), 58 binding the S2 domain, and 19 binding

15 an indeterminant epitope or failing to recognize spike in a MSD binding assay (Figure 1B). Among

these, 4 RBD targeting antibodies, A19-46.1, A19-61.1, A23-58.1 and B1-182.1, were shown to

have especially potent pseudovirus neutralization (IC50 0.0025-0.0709 µg/mL) (Figure 1C, E).

Live virus neutralization (17) revealed similar high potent neutralization by all four antibodies

(IC50 0.0021-0.0048 µg/mL) (Figure 1D-E). All antibody Fabs exhibited nanomolar affinity for

20 SARS-CoV-2 S-2P (i.e., 2.3-7.3 nM), consistent with their potent neutralization (Figure 1E).

Since VOCs have been reported to contain mutations that confer resistance to RBD-

directed therapeutic antibodies such as LY-CoV555 (18–20), we examined whether the epitopes

targeted by the four high-potency antibodies were distinct from LY-CoV555. We used a biolayer

interferometry-based (BLI) competition binding assay to compare the binding profile of these

antibodies to LY-CoV555. We noted that while LY-CoV555 prevented the binding of each of

the experimental antibodies, the block was not bidirectional; the experimental antibodies did not

impact the binding of LY-CoV555. This suggests that these antibodies bind distinct epitopes

5 from LY-CoV555 (Figure 1F). We found that A23-58.1 and B1-182.1 exhibit similar binding

profiles and that A19-61.1 and A19-46.1 likewise display a shared binding pattern. However, the

latter two antibodies can be distinguished from each other by their capacity to compete for

binding with the RBD-targeting antibody S309 (21) (Figure 1F). S309 binds an epitope in RBD

that is accessible in the up or down position but does not compete with the SARS-CoV-2

10 receptor protein, angiotensin-converting enzyme (ACE2), and is a Class III RBD antibody (22).

To further classify the antibodies, we examined whether these antibodies prevent the binding of

ACE2 to spike proteins. We noted that in both BLI-competition and cell surface binding assays,

all four experimental antibodies prevented the binding of ACE2 to spike (Figure 1F, Figure S2).

This suggests that A19-46.1, A23-58.1 and B1-182.1 neutralize infection by blocking the

15 interaction of RBD with ACE2 and would be classified as either Class I (i.e., ACE2 blocking,

binding RBD up only) or II (i.e., ACE2 blocking, binding RBD up or down) RBD antibodies

(22). A19-61.1 competes with S309 and blocks ACE2 binding suggesting that it may sterically

block ACE2 binding similar to the Class III antibody REGN10987. To refine the classification of

these antibodies, we performed negative stain 3D reconstruction and found that A19-46.1 and

20 A19-61.1 bound near one another with RBD in the down position (Figure 1G), consistent with

them being Class II and Class III antibodies, respectively. Similarly, A23-58.1 and B1-182.1

bound to overlapping regions when RBD is in the up position, suggesting that they are Class I

antibodies.

Because each donor subject was infected with ancestral WA-1 variants, we evaluated

antibody activity against recently emerged variants like D614G, which has become the dominant

variant across the world (23). We observed that, similar to LY-CoV555, neutralization potency

was increased against D614G compared to WA-1, with the IC50 and IC80 of each experimental

5 antibody 1.4 to 6.3-fold lower than that seen for the WA-1 (IC50 of 0.0008-0.0203 µg/mL and IC80

of 0.0026-0.0435 µg/mL) (Figure 2A,C, Figure S3).

Next, we assessed antibody binding to eleven cell surface expressed spike variants that

have appeared subsequent to D614G (6–9, 23). We found that all control and experimental

antibodies showed a minor reduction in binding (<2-fold) to Var3 (N439K/D614G). Despite this,

10 their neutralization capacities were not significantly impacted, with the exception of REGN10987

(2.00 µg/mL) as reported previously (24) (Figure 2A-C, Figure S3). We also noted that while none

of the experimental antibodies showed large reductions in binding, LY-CoV555, CB6 (25) and

REGN10933 (26) each showed significant (>10-fold) binding deficits to one or more variants (i.e.,

Var4, Var9, Var10 or Var11) in these cell-based binding assays (Figure 2A,B, Figure S3).

15 Given the impact on antibody-spike binding, we evaluated the capacity of each antibody

to neutralize virus particles pseudotyped with variant spike proteins containing one to three

mutations on the D614G backbone (i.e., D614G, Var2-7 and Var10-11). Consistent with published

data, REGN10933 did not neutralize Var4 (Y453F/D614G) or Var11

(K417N/E484K/N501Y/D614G) (20, 27, 28); CB6 did not neutralize Var11; and LY-CoV555,

20 REGN10989, and REGN109333 showed significant potency reductions (28-fold to knockout) for

neutralization of viruses containing E484K (20, 28) (Figure 2C). For the experimental antibodies,

with the exceptions of Var5 (N501Y/D614G) and Var10 (E484K/D614G), neutralization by A23-

58.1 was not significantly altered. For Var5, the IC50 of A23-58.1 was 5-fold lower (IC50 <0.0006

µg/mL) (Figure 2C). In contrast, for Var10, the IC50 was increased 4-fold relative to the ancestral

WA-1 but was still highly potent at 0.0102 µg/mL. Neutralization by B1-182.1 maintained high-

potency (IC50 <0.0032 µg/mL) for all variants and showed more than 4-fold improved potency for

6 of the 10 variants tested (IC50 <0.0008 µg/mL) (Figure 2C). Neutralization of all variants (i.e.,

5 Var2-7, Var10-11) by A19-61.1 was 3 to 6-fold more potent than WA-1 (WA-1 IC50

0.0709 µg/mL; variants IC50 0.0111-0.0237 µg/mL) (Figure 2A,C). Finally, neutralization by A19-

46.1 was similar to WA-1 for all variants except Var10 and Var11, which were still highly potent

despite having IC50 values that were 2 to 3-fold less active (Var10: 0.095; Var11: 0.0618; WA-1:

0.0398 µg/mL) (Figure 2C). Together, these data show the capacity of these newly identified

10 antibodies to maintain high neutralization potency against a diverse panel of variant spike proteins.

In addition, we analyzed neutralization against two widely circulating, dominant virus

variants with high-transmissibility, B.1.1.7 (a.k.a., UK VOC2020/12/01) (6, 11) and two versions

of the B.1.351 variant (a.k.a., SA 501Y.V2), differing by the presence (B.1.351.v1) or absence

(B.1.351.v2) of R246I (7)(Figure 2A,D). Consistent with published data, we found that LY-

15 CoV555, CB6, REGN10989, REGN10933 and REGN10987 maintained high potency against

B.1.1.7 (IC50 0.0001-0.0401 µg/mL) but LY-CoV555, CB6, REGN10989 and REGN10933 were

not able to neutralize either B.1.351 variant (IC50 >10 µg/mL) (Figure 2) (20, 27, 28). In

comparison, A23-58.1, B1-182.1, A19-46.1 and A19-61.1 maintained similar or improved potency

(IC50 <0.0006-0.0115 µg/mL) against B.1.1.7 relative to WA-1. A19-46.1 maintained potency

20 against B.1.351.v2 and ~2.5-fold lower neutralization against B.1.351.v1 (WA-1 IC50 0.0398

µg/mL; B.1.351.v1 IC50 0.1014 µg/mL; B.1.351.v2 IC50 0.0571 µg/mL), while A23-58.1, B1-

182.1 and A19-61.1 maintained high potency against both B.1.351 variants (IC50 <0.0006-0.0108

µg/mL) (Figure 2C). These results indicate that despite being isolated from subjects infected with

early ancestral SARS-CoV-2 viruses, these antibodies have high-potency against B.1.1.7 and

B.1.351 VOCs.

The two most potent antibodies, A23-58.1 and B1-182.1, shared highly similar gene family

usage in their heavy and light chains, despite being from different donors (Table S1). Both use

5 IGHV1-58 heavy chains and IGKV3-20/IGKJ1 light chains. This antibody gene family

combination has previously been noted to be present in other COVID-19 convalescent subjects

and has been proposed as a public clonotype (29–32). To gain structural insights on the interaction

between this class of antibodies and the SARS-CoV-2 spike, we mixed A23-58.1 Fab and spike at

a molar ratio of 3.6:1 and purified the complex by size-exclusion chromatography. We collected

10 single particle cryo-EM data on a Titian Krios and determined the structure of the complex at 3.39

Å resolution (Figure 3A, Figure S4 and Table S2) and revealed that the antibody bound to spike

with all RBDs in the up position, confirming the negative stain results (Figure 3A, Figure 1G).

However, the cryo-EM reconstruction density of the RBD and A23-58.1 interface was poor due to

conformational variation.

15 To resolve the antibody-antigen interface, we performed local refinement and improved

the local resolution to 3.89 Å which enabled detailed analysis of the mode of antibody recognition

(Figure S4). Antibody A23-58.1 bound to an epitope on the RBD that faces the 3-fold axis of the

spike and is accessible only in the RBD-up conformation (Figure 3A). The interaction buried a

total of 601 Å2 surface area from the antibody and 607 Å2 from the spike (Table S3). The A23-

20 58.1 paratope constituted all six complementarity-determining regions (CDR) with both heavy

chain and light chain contributing 73% and 27% of binding surface area, respectively (Figure 3B,

Figure 3C and Table S3). The 14-residue-long CDR H3, which provided 48% of the heavy chain

paratope, kinked at Pro95 and Phe100F (Kabat numbering scheme for antibody residues) to form

a foot-like loop that is stabilized by an intra-loop disulfide bond between Cys97 and Cys100B at

the arch. A glycan was observed to attach the CDR H3 Asn96 (Figure S4F). The CDRs formed an

interfacial crater with a depth of ~10 Å and a diameter of ~20 Å at the opening. Paratope residues

inside the crater were primarily aromatic or hydrophobic. With CDR H3 Pro95 and Phe100F

5 paving the bottom, CDR H1 Ala33, CDR H2 Trp50 and Val52, and CDR H3 Val100A lined the

heavy chain side of the crater (Figure 3B/C). On the light chain side, CDR L1 Tyr32 and CDR L3

residues Tyr91 and Trp96 provided 80% of the light chain binding surface (Figure 3B,C). In

contrast, paratope residues at the rim of the crater are mainly hydrophilic, for example, Asp100D

formed hydrogen bonds with Ser477 and Asn487 of the RBD (Figure 3B-C, Table S3).

10 The A23-58.1 epitope is composed of residues between b5 and b6 at the tip of RBD (Figure

3B and 3D). With the protruding Phe486 dipping into the crater formed by the CDRs, these

residues formed a hook-like motif that is stabilized by an intra-loop disulfide bond between

Cys480 and Cys488. Aromatic epitope residues, including Phe456, Tyr473, Phe486 and Tyr489,

provided 38% of the binding surface (237.5 Å2) (Figure 3B and 3D, Table S3). Lys417 and Glu484,

15 which are located at the outer edge of the epitope, contributed only 3.7% of the binding surface

(Figure 3B and Table S3). Overall, the cryo-EM analysis provided structural basis for the potent

neutralization of the E484K mutant by A23-58.1. The binding mode of A23-58.1 is very similar

to that of a previously reported IGHV1-58/IGKV3-20-derived antibody, S2E12 (29) confirming

that they are members of the same structural class (Figure 3C and 3D). In addition, sequence

20 analysis indicates that B1-182.1 is likely also a member of this class – and thus shares the same

mode of recognition. In fact, B1-182.1 share a nearly identical IGHD2-15-derived CDR H3

sequence with S2E12 (Figure 3C). The lack of impact of emerging resistance mutations on B1-

182.1 can be explained by the same mechanism whereby A23-58.1 antibody also is not impacted

by these variants. Interestingly, while A23-58.1 and B1-182.1 both have 10-residue-long CDR

L3s, S2E12 includes the insertion of Val91A (Figure 3C) and the longer CDR L3 may therefore

lead to differences in binding/neutralization capacity for viruses with mutations at the Glu484

location where CDR L3 contacts.

5 We next sought to understand the structural mechanisms by which A23-58.1 overcomes

mutations that cause reduced antibody potency against virus variants. To do this, we superposed

the antibody-RBD complex structures of CB6 (PDB ID 7C01) (25), REGN10933 (PDB ID 6XDG)

(26, 27) and LY-CoV555 (PDB ID 7KMG) (18) with the A23-58.1 structure over the RDB region.

Both REGN10933 and CB6 bind to the same side of the RBD that A23-58.1 contacted (Figure 3D

10 and 3E). However, their binding surfaces were all shifted towards the saddle of the open RBD and

encircled residues Lys417, Tyr453, Glu484 and Asn501 within the epitope (Figure 3D and 3E),

mutations K417N and Y453F potently abolished key interactions and led to the loss of

neutralization for both REGN10933 and CB6 (Figure 2). In contrast, LY-CoV555 approached the

RBD from a different angle with its epitope centered around Glu484 (Figure 3D and 3F). Modeling

15 indicated that mutation E484K may abolish key interactions with Arg50 and Arg96 of LY-CoV555

and cause a clash with CDR H3 of LY-CoV555. These structural data suggest that the unique

binding modes of A23-58.1 and potentially B1-182.1 derived from the same germline enabled

their high effectiveness against the new SARS-CoV-2 variants.

We next used the structural analysis to investigate the relative contribution of predicted

20 contact residues on binding and neutralization (Figure 3D). Cell surface expressed spike binding

by A23-58.1 and B1-182.1 were knocked out by F486R, N487R, and Y489R (Figure 4A, Figure

S5), resulting in a lack of neutralization for viruses pseudotyped with spikes containing these

mutations (Figure 4B). In contrast, binding and neutralization of A19-46.1 and A19-61.1 were

minimally impacted by these changes (Figure 4A,B, Figure S5). CB6, LY-CoV555 and

REGN10933 binding and neutralization were also impacted by the three mutations, consistent with

the structural analysis that these residues are commonly shared contact(s) among the impacted

antibodies. Taken together, the shared binding and neutralization defect imposed by these

5 mutations on A23-58.1 and B1-182.1 suggests that the hook-like motif and CDR crater are critical

for the binding of antibodies within the VH1-58 public class.

Next, based on structural modeling of the negative stain EM density (Figure 1G), we chose

several mutants to investigate the determinants of binding for A19-46.1 and A19-61.1. Under

conditions where A23-58.1 and B1-182.1 were not impacted, we found that L452R, F490R and

10 S494R knocked out binding for A19-46.1 and S494R knocked out binding for A19-61.1 (Figure

4A, Figure S5). In addition, the partial overlap of susceptibility to the selected mutations between

A19-46.1 and A19-61.1 is in agreement with the antibody competition data showing similar, but

distinct profiles (Figure 1F) and indicates that these antibodies represent distinct antibody classes.

To explore resistance mechanisms that might be generated during the course of infection,

15 we applied antibody selection pressure to replication competent vesicular stomatitis virus (rcVSV)

expressing the WA-1 SARS-CoV-2 spike (rcVSV-SARS2) (33) to identify spike mutations that

confer in vitro resistance against A23-58.1, B1-182.1, A19-46.1 or A19-61.1 (Figure S6). rcVSV-

SARS2 was incubated with increasing concentrations of antibody, and cultures from the highest

concentration of antibody with >20% cytopathic effect (CPE) were carried forward into a second

20 round of selection to drive resistance (27)(Figure S6). A shift to higher antibody concentrations

required for neutralization indicated the presence of resistant viruses. To define and determine the

relative frequency of mutations that accumulated at positions within the spike of resistant viruses,

we performed Illumina-based shotgun sequencing (Figure S6). Variants present at a frequency of

greater than 10% and increasing from round 1 to round 2 were considered to be positively selected

resistant viruses. For A19-46.1, three selection mutations were generated: Y449S (freq. 15%),

N450S (freq. 16%) and F490V (freq. 58%) (Figure 4C, Figure S7). The most dominant, F490V,

was found in 58% of sequences and is consistent with the previous finding that F490R knocked

5 out binding and neutralization of A19-46.1 (Figure 4A,C). These residues are clustered near one

another on RBD and would be expected to be accessible when RBD is in the up or down position

(Figure S7). Several of these contacts are shared by Class II RBD antibodies (22, 34) and

REGN10933 (26, 35). Despite these shared contacts, A19-46.1 is able to neutralize variants that

REGN10933 cannot (i.e., Var10, Var11 and B.1.351) (Figure 2A-C), indicating that A19-46.1

10 makes critical contacts that are able to overcome resistance mutations that affect REGN10933.

Three residues were positively selected in the presence of A19-61.1: K444E (freq. 57%),

G446V (freq. 24%) and G593R (freq. 19%) (Figure 4A) and did not overlap with those selected

by A19-46.1. G593R is located outside the RBD domain and the others are clustered nearby

S494R, identified previously (Figure 4A, Figure S7). The highest frequency change was at K444

15 and represented 57% of the sequences. This residue has been shown to be critical for the binding

of Class III RBD antibodies such as REGN10987 (22, 26, 27, 35). Taken together with the ability

of A19-61.1 to block ACE2 binding (Figure 1F, Figure S2) and differential neutralization between

the Class III antibody REGN10987 and A19-61.1 against Var3 (N439K/D614G) (i.e., significantly

reduced neutralization with REGN10987) (Figure 2C), A19-61.1 likely targets an epitope distinct

20 epitope from REGN10987 and other Class III RBD antibodies.

Finally, a single mutation, F486S (freq. 91%) was positively selected for when virus was

incubated in the presence of A23-58.1. This is in agreement with our structural analysis (Figure

3B) that showed that F486 is located at the tip of RBD “hook” and contributes to the binding

interface in the antibody “crater”. This is dominated by aromatic side chains that form the “hook”

and “crater” interface (Figure 3A,B). Therefore, one possible explanation for the loss in activity is

through the replacement of a hydrophobic aromatic residue (i.e., phenylalanine) with a small polar

side chain (i.e., serine) (Figure 3C).

5 To probe the relevance of in vitro derived resistance variants to the potential for clinical

resistance we next examined spikes derived from circulating virus variants. We investigated the

relative frequency of variants containing the mutations present in the GISAID sequence database

using the COVID-19 Viral Genome Analysis Pipeline (cov.lanl.gov)(23) in which, as of February

11, 2021, there were 417,702 entries. Out of these, the original WA-1 residues critical for A19-

10 46.1 activity (i.e., Y449, N450, L452, F490 and S494) were present in 99.81-99.99% of sequences

available. Of the residues identified in our experiments to mediate resistance to A19-46.1, Y449S,

N450S, L452R, F490L/V and S494R, only F490V has been noted in the database (5 sequences,

0.001%) (Figure 4, Figure S7). For A19-61.1, the ancestral WA-1 residues, K444, G446, S494 and

G593, were present in 99.81-100% of entries. Of the resistance-inducing residues identified, i.e.,

15 K444E, G446V, S494R, and G593R, only G446V has been noted in the database (106 sequences,

0.03%) (Figure 4, Figure S7). Finally, for A23-58.1 and B1-182.1 the ancestral WA-1 residues

F486, N487 and Y489 were present in >99.99% of sequences and none of the binding/resistance

mutations identified in our experiments were noted in the database. The relative lack of resistance

mutations found in circulating viruses suggests that the in vitro derived mutations exact a fitness

20 cost on the virus and are not tolerated during infection but could also reflect either under-sampling

or the absence of other sources of selection pressure.

Viral genome sequencing has suggested the possibility that in addition to spread via

transmission, convergent selection of de novo mutations may be occurring(6–9, 13, 23, 36).

Therefore, effective therapeutic antibody approaches might require new antibodies or

combinations of antibodies to mitigate the impact of mutations. Based on their complementary

modes of spike recognition and breadth of neutralizing activity, we hypothesized that combination

of B1-182.1 with either A19-46.1 or A19-61.1 would decrease the rate of in vitro resistance

5 acquisition compared to each antibody alone. As a first test, we used negative stain EM 3D

reconstructions to determine whether these combinations are able to bind simultaneously to spike

protein. Consistent with the competition data (Figure 1F), we found that the Fabs in both

combinations were able to engage spike simultaneously with RBD in the up position (Figure 4D).

Furthermore, we noted that binding was in a 3:1 Fab:spike ratio in most of the observed particles

10 (Figure 4D), revealing that the epitopes of A19-46.1 and A19-61.1 on the spike are accessible in

both RBD up and down positions (Figure 1G and Figure 4D). This suggests that the combination

allows alternative preferential mode of RBD engagement (i.e., RBD up vs. RBD down) by A19-

46.1 and A19-61.1 that is not seen in the absence of B1-182.1 or A23-58.1.

Next, we determined the rates of in vitro resistance acquisition of combined treatments

15 compared to individual antibodies using an rcVSV SARS-CoV-2 resistance generation approach.

We evaluated the capacity of individual antibodies or combinations to prevent the appearance of

rcVSV SARS –CoV-2-induced cytopathic effect (CPE) throughout multiple rounds of passaging

in the presence of increasing concentrations of antibodies. In each round, the well with the highest

concentration of antibody with at least 20% CPE was carried forward into the next round. We

20 found that wells with A19-61.1 or A19-46.1 single antibody treatment reached the 20% CPE

threshold in their 50 µg/mL well after 3 rounds of selection (Figure 4E). Similarly, B1-182.1 single

antibody treatment reached >20% CPE in the 50 µg/mL wells after 4 rounds (Figure 4E).

Conversely, for both dual treatments (i.e., B1-182.1/A19-46.1 or B1-182.1/A19-61.1) the 20%

CPE threshold was reached only at a concentration of 0.08 µg/mL and did not progress to higher

concentrations despite 5 rounds of passaging (Figure 4E). While further data are required, these

results suggest that such combinations may lower the risk that a natural variant will lead to the

complete loss of neutralizing activity and suggests a path forward for these antibodies as

5 combination therapies.

Worldwide genomic sequencing has revealed the occurrence of SARS-CoV-2 variants that

increase transmissibility and reduce potency of vaccine-induced and therapeutic antibodies (10–

16). Recently, there has been a significant concern that antibody responses to natural infection and

vaccinations using ancestral spike sequences may have focused responses that are overcome by

10 mutations present in more recent isolates (e.g., E484K in B.1.351) (12–16). As a first step to

address the risk of reduced antibody potency against new variants, we isolated and defined new

antibodies with neutralization breadth covering newly emerging SARS-CoV-2 variants. Increased

potency and breadth were mediated by binding to regions of the RBD tip that are offset from

E484K, which is a major determinant of resistance in VOCs (12–16). Our results show that highly

15 potent neutralizing antibodies with activity against these variants was present in at least 3 of four

convalescent subjects who had been infected with ancestral variants of SARS-CoV-2.

Furthermore, two antibodies from different subjects used VH genes associated with previously

described public clonotypes (29, 30). Overall, these data establish the rationale for a vaccine

boosting regimen that may be used to selectively induce immune responses that increase the

20 breadth and potency of antibodies targeting the RBD region of the spike glycoprotein.

Furthermore, since both variant sequence analysis and in vitro time to escape experiments suggest

that combinations of these antibodies may have a lower risk for loss of neutralizing activity, these

antibodies represent a potential means to achieve both breadth against current VOCs and to

mitigate risk against those that may develop in the future.

5 References and Notes:

1. COVID-19 Map - Johns Hopkins Coronavirus Resource Center, (available at

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Acknowledgments: We would like to thank the staff of the Clinical Trials Program of the

5 Vaccine Research Center and the volunteers that made this research possible. We also appreciate

the assistance of Dr. Ruth Hunegnaw for assistance with figure preparation. We are grateful to

Tara L. Fox of NCEF for collecting cryo-EM data and for technical assistance with cryo-EM

data processing. We would like to thank Avan Antia, Rachel L. Davis and Farida Laboune for

technical assistance with sequencing.

Funding: This work was funded by the intramural research program of the Vaccine Research

Center, NIAID, NIH. Funding was also supported by the North Carolina Policy Collaboratory at

the University of North Carolina at Chapel Hill with funding from the North Carolina

Coronavirus Relief Fund established and appropriated by the North Carolina General Assembly.

15 David R. Martinez is funded by a Burroughs Wellcome Fund Postdoctoral Enrichment Program

Award, a Hanna H. Gray Fellowship from the Howard Hughes Medical Institute, and was

supported by an NIH NIAID T32 AI007151 and an NIH F32 AI152296. Additional support for

this work was provided by Federal funds from the Frederick National Laboratory for Cancer

Research under Contract HHSN261200800001E (Y.T.). Cryo-EM data was collected at the

20 National CryoEM Facility (NCEF) of the National Cancer Institute. This research was, in part,

supported by the National Cancer Institute’s National Cryo-EM Facility at the Frederick National

Laboratory for Cancer Research under contract HSSN261200800001E.

Author contributions: T.R., E.P., A.D., L.N., N.D.R., R.M., J.M., C.S., L.W., K.C. and E.C.

designed and performed cell sorting experiments. A.R., S.D. and C.S. performed and analyzed

sequencing data. Proteins, antibody and other reagents were produced by W.S., I.T., L.W., T.Z.,

5 A.O., E.P., T.R., J.M., O.A., L.C., A.D., E.S.Y. Y.Z., B.Z., A.N. and T.L., J.M., L.W., T.Z.,

Y.Z., W.S., E.S.Y., A.P., O.O., A.R., C.S., S.D., S.N., C.H., D.M., C.T., C.S. and D.W.

conceived of, designed experiments, performed experiments, data analysis and reporting. M.G.,

A.W., L.N. and I.G. for research subject recruitment, collection of samples and maintenance of

the sample repository. T.Z. and Y.T. led negative stain electron microscopy and cryo-EM

10 studies. J.M., N.J.S., J.R.M., D.D., B.S.G, A.M., P.K. and R.S.B. supervised experiments. J.M.,

N.J.S., T.Z., L.W. and C.S. wrote the manuscript with help from all authors.

Competing interests: J.M., L.W., C.M., J.R.M, D.D, N.J.S., A.R., T.Z., P.K., W.S., Y.Z.,

E.S.Y., M.R., R.M. and A.P. are inventors on US patent application No. 63/147,419.

Data and materials availability: All data is available in the main text or the supplementary

15 materials. Atomic coordinates and cryo-EM maps of the reported structure have been deposited

into the Protein Data Bank and Electron Microscopy Data Bank under the session codes PDB

7LRT and EMD-23499 for SARS-CoV-2 spike in complex with antibody A23-58.1, and PDB

7LRS and EMD-23498 for local refinement of the RDB-antibody A23-58.1 region. Plasmids are

available from N.J.S. under a materials transfer agreement with the National Institutes of Health.

20 Supplementary Materials:

Materials and Methods

Figures S1-S7

Tables S1-S3

References (36-52)

Fig. 1. Identification and classification of highly potent antibodies from convalescent

SARS-CoV-2 subjects.

(A) Final flow cytometry sorting gate of CD19+/CD20+/IgG+ or IgA+ PBMCs for four

5 convalescent subjects (Subjects 1-4). Shown is the staining for RBD-SD1 BV421, S1 BV786 and

S-2P APC or Ax647. Cells were sorted using indicated sorting gate (pink) and percent positive

cells that were either RBD-SD1, S1 or S-2P positive is shown for each subject.

(B) Gross binding epitope distribution was determined using an MSD-based ELISA testing

against RBD, NTD, S1, S-2P or HexaPro. S2 binding was inferred by S-2P or HexaPro binding

10 without binding to other antigens. Indeterminant epitopes showed a mixed binding profile. Total

number of antibodies (i.e., 200) and absolute number of antibodies within each group is shown.

capacity of the indicated antibodies (n=3).

(D) Live virus neutralization assay for A23-58.1 (n=2), A19-46.1 (n=2), A19-61.1 (n=2) and B1-

15 182.1 (n=3).

(E) Table showing antibody binding target, IC50 for pseudovirus and live virus neutralization and

Fab:S-2P binding kinetics (n=2) for the indicated antibodies.

(F) Biolayer interferometry-based epitope binning experiment. Competitor antibody (y-axis) is

bound to S-2P prior to incubation with the analyte antibody or ACE2 protein (x-axis) as

20 indicated and percent competition range bins are shown as red (>=75%), orange (60-75%) or

white <60%) (n=2). mAb114 is an anti-Ebola glycoprotein antibody and is included as a negative

control (37)

(G) Negative stain 3D reconstructions of SARS-CoV-2 spike and Fab complexes. A19-46.1 and

A19-61.1 bind to RBD in the down position while A23-58.1 and B1-182.1 bind to RBD in the up

position. Representative classes were shown with 2 Fabs bound, though stoichiometry at 1 to 3

were observed.

5 Fig. 2. Neutralization and binding activity against spike proteins from circulating variants.

(A) Table showing domain and mutations relative to WA-1 for each of the 13 variants tested in

panels B-C.

(B) Spike protein variants were expressed on the surface of HEK293T cells and binding to the

indicated antibody was measured using flow cytometry. Data is shown as Mean Fluorescence

10 intensity (MFI) normalized to the MFI for the same antibody against the D614G parental variant.

Percent change is indicated by a color gradient from red (increased binding, Max 250%) to white

(no change, 100%) to blue (no binding, 0%). Black indicates binding was not tested.

in (A). Ranges are indicated by colors white (>10 µg/mL), light blue (1-10 µg/mL), yellow (0.1-

15 1 µg/mL), orange (0.05-0.1 µg/mL), red (0.01-0.05 µg/mL), maroon (0.001-0.01 µg/mL) and

purple (<0.001 µg/mL). Black indicates a variant that was not tested.

(D) Location of spike protein variant mutations on the spike glycoprotein for B.1.1.7 (left) and

B.1.351 (right). P681H is not resolved in the structure and therefore its location is not noted in

B.1.1.7.

Fig. 3. Structural basis of A23-58.1 binding.

(A) Cryo-EM structure of A23-58.1 Fab in complex with SARS-CoV-2 HexaPro spike. Overall

density map is shown to the left with protomers colored light green, gray and cyan. One of the

A23-58.1 Fab bound to the RBD is shown in orange and blue. Structure of the RBD and A23-

58.1 after local focused refinement was shown to the right. The heavy chain CDRs are colored

brown, salmon and orange for CDR H1, CDR H2 and CDR H3, respectively. The light chain

CDRs are colored marine blue, light blue and purple blue for CDR L1, CDR L2 and CDR L3,

5 respectively. The contour level of Cryo-EM map is 5.7s.

(B) Interaction between A23-58.1 and RBD. All CDRs were involved in binding of RBD.

Epitope of A23-58.1 is shown in bright green surface with a yellow border (left, viewing from

antibody to RBD). RBD mutations in current circulating SARS-CoV-2 variants are colored red.

Lys417 and Glu484 are located at the edge of the epitope. The tip of the RBD binds to a cavity

10 formed by the CDRs (right, viewing down to the cavity). Interactions between

aromatic/hydrophobic residues are prominent at the lower part of the cavity. Hydrogen bonds at

the rim of the cavity are marked with dashed lines. RBD residues were labeled with italicized

font.

15 underlined. Paratope residues for A23-58.1 and S2E12 were highlighted in green and light

brown, respectively.

(D) Epitope of A23-58.1 on RBD. Epitope residues for different RDB-targeting antibodies are

marked with * under the RBD sequence.

(E) Comparison of binding modes of A23-58.1 and REGN10933. One Fab is shown to bind to

20 the RBD on the spike. The shift of the binding site to the saddle of RBD encircled Lys417,

Glu484 and Tyr453 inside the REGN10933 epitope (violet), explaining its sensitivity to the

K417N, Y453F and E484K mutations.

(F) Comparison of binding modes of A23-58.1 and LY-CoV555. One Fab is shown to bind to

the RBD on the spike. Glu484 is located in the middle of LY-CoV555 epitope (light orange),

explaining its sensitivity to the E484K mutation.

5 Fig. 4. Critical binding residue determination and mitigation of escape risk using dual

antibody combinations

(A) The indicated Spike protein mutations predicted by structural analysis were expressed on the

surface of HEK293T cells and binding to the indicated antibody was measured using flow

cytometry. Data is shown as Mean Fluorescence intensity (MFI) normalized to the MFI for the

10 same antibody against the WA-1 parental binding. Percent change is indicated by a color

gradient from red (increased binding, Max 250%) to white (no change, 100%) to blue (no

binding, 0%).

(B) IC50 and IC80 values for the indicated antibodies against WA-1 and the 10 mutations. Ranges

are indicated by colors white (>10 µg/mL), light blue (1-10 µg/mL), yellow (0.1-1 µg/mL),

15 orange (0.05-0.1 µg/mL), red (0.01-0.05 µg/mL), maroon (0.001-0.01 µg/mL) and purple

(<0.001 µg/mL).

CoV-2 WA-1 was incubated with serial dilutions of the indicated antibodies and wells with

cytopathic effect (CPE) were passaged forward into subsequent rounds (Figure S6) after 48-72

20 hours. Total supernatant RNA was harvested and viral genomes shotgun sequenced to determine

the frequency of amino acid changes. Shown are the spike protein amino acid/position change

and frequency as a logo plot.

(D) Negative stain 3D reconstruction of the ternary complex of spike with Fab B1-182.1 and

A19-46.1 (left) or A19-61.1 (right).

(E) rcVSV SARS-CoV-2 was incubated with increasing concentrations (1.3e-4 to 50 µg/mL) of

either single antibodies (A19-46.1, A19-61.1 and B1-182.1) and combinations of antibodies (B1-

5 182.1/A19-46.1 and B1-182.1/A19-61.1). Every 3 days, wells were assessed for CPE and the

highest concentration well with the >20% CPE was passaged forward onto fresh cells and

antibody containing media. Shown is the maximum concentration with >20% CPE for each of

the test conditions in each round of selection. Once 50 µg/mL has been reached, virus was no

longer passaged forward and a dashed line is used to indicate maximum antibody concentration

10 was reached in subsequent rounds.

bioRxiv preprint doi: https://doi.org/10.1101/2021.02.25.432969; this version posted March 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license.

Figure 1

A Subject Antigen Probe Sort BCEpitope Pseudovirion D Live virus 5 5 10 10 Subject 1 Subject 2 Distribution Neutralization Neutralization

4 0.65% 4 0.621% 100 100 10 10 9 10 80 80 3 3 LY-COV555 10 10 60 B1-182.1 60 B1-182.1 A23-58.1 A23-58.1 2 2 40 40 10 10 58 200 77 A19-46.1 A19-46.1 0

0 RBD-SD1 BV421 RBD-SD1 BV421 20 A19-61.1 20 A19-61.1

3 4 5 3 4 5 % Neutralizatio n 0 10 10 10 0 10 10 10 % Neutralizatio n 0 0 -4 -2 0 2 -4 -2 0 2 S-2P APC S-2P APC 46 10 10 10 10 10 10 10 10 5 5 Antibody Concentration [μg/mL] Antibody Concentration [μg/mL] 10 10 Subject 3 Subject 4 4 4 10 2.96% 10 0.402% RBD NTD S2 E Pseudovirus Neut. Live Virus Neut. S-2P binding kinetics 3 3 10 10 indeterminant mAb Target IC50 (ug/mL) IC50 (ug/mL) KD (nM) kon (1/Ms) koff (1/s) A19-46.1 RBD 0.0398 0.0048 3.58 3.79 e5 1.35 e-3

2 2 A19-61.1 RBD 0.0709 0.0022 2.33 3.04 e5 7.06 e-4

10 S1 BV786 10 no binding A23-58.1 RBD 0.0025 0.0021 7.3 7.13 e5 5.20 e-3 0 0 RBD-SD1 BV421 B1-182.1 RBD 0.0034 0.0024 2.55 8.65 e5 2.21 e-3 3 4 5 3 4 5 0 10 10 10 0 10 10 10 F S-2P APC S-2P AX647 G Analyte A19-46.1 A19-61.1 A23-58.1 B1-182.1

A19- A19- LY-CoV A23- B1- S309 ACE2 61.1 46.1 555 58.1 182.1 Fab Fab Fab Fab Fab Fab Fab Fab S309

A19- 61.1

A19- 46.1

LY-CoV Spike Spike Spike 555

A23- 58.1 Competitor

B1- 182.1

mAb 114

% Competition >75% 60-75% < 60%

Fig. 1. Identification and classification of highly potent antibodies from convalescent SARS-CoV-2 subjects.

(A) Final flow cytometry sorting gate of CD19+/CD20+/IgG+ or IgA+ PBMCs for four convalescent subjects (Subjects 1-4). Shown is the staining for RBD-SD1 BV421, S1 BV786 and S-2P APC or Ax647. Cells were sorted using indicated sorting gate (pink) and percent positive cells that were either RBD-SD1, S1 or S-2P positive is shown for each subject. (B) Gross binding epitope distribution was determined using an MSD-based ELISA testing against RBD, NTD, S1, S-2P or HexaPro. S2 binding was inferred by S-2P or HexaPro binding without binding to other antigens. Indeterminant epitopes showed a mixed binding profile. Total number of antibodies (i.e., 200) and absolute number of antibodies within each group is shown. (C) Lentivirus particles pseudotyped with WA-1 spike were used to test the neutralization capacity of the indicated antibodies (n=3). (D) Live virus neutralization assay for A23-58.1 (n=2), A19-46.1 (n=2), A19-61.1 (n=2) and B1-182.1 (n=3). (E) Table showing antibody binding target, IC50 for pseudovirus and live virus neutralization and Fab:S-2P binding kinetics (n=2) for the indicated antibodies. (F) Biolayer interferometry-based epitope binning experiment. Competitor antibody (y-axis) is bound to S-2P prior to incubation with the analyte antibody or ACE2 protein (x-axis) as indicated and percent competition range bins are shown as red (>=75%), orange (60-75%) or white <60%) (n=2). mAb114 is an anti-Ebola glycoprotein antibody and is included as a negative control (37) (G) Negative stain 3D reconstructions of SARS-CoV-2 spike and Fab complexes. A19-46.1 and A19-61.1 bind to RBD in the down position while A23-58.1 and B1-182.1 bind to RBD in the up position. Representative classes were shown with 2 Fabs bound, though stoichiometry at 1 to 3 were observed. bioRxiv preprint doi: https://doi.org/10.1101/2021.02.25.432969; this version posted March 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license.

Figure 2

B.1.351 B.1.351 Domain Mutations D614G Var2 Var3 Var4 Var5 Var6 Var7 B.1.1.7 Var9 Var10 Var11 A .v2 .v1 L18F/D80A/D215G/ L242-244 NTD x 5 NTD x 5 H69 /V70 H69-70 H69-70 H69-70 NTD Y144 Y144 A222V A222V R246I R246I K417N K417N K417N K417N K417N N439K N439K RBD Y453F Y453F E484K E484K E484K E484K E484K N501Y N501Y N501Y N501Y N501Y N501Y N501Y A570D A570D S1-C-term D614G D614G D614G D614G D614G D614G D614G D614G D614G D614G D614G D614G D614G D614G P681H P681H T716I/S982A/D1118H S2 x 3 S2 A701V A701V A701V

B B.1.351 B.1.351 D614G Var2 Var3 Var4 Var5 Var6 Var7 B.1.1.7 Var9 Var10 Var11 Cell Surface Binding .v2 .v1 MFI Normalized A23-58.1 to 614G B1-182.1 250% A19-61.1 200% A19-46.1 150% LY-COV555 100% CB6

50% REGN10987

0% REGN10933

B.1.351 B.1.351 C WA-1 D614G Var2 Var3 Var4 Var5 Var6 Var7 B.1.1.7 Var9 Var10 Var11 .v2 .v1 A23-58.1 0.0025 0.0018 0.0020 0.0031 0.0011 <0.0006 0.0017 0.0009 <0.0006 0.0102 0.0058 0.0016 0.0015 B1-182.1 0.0034 0.0008 0.0008 0.0011 0.0007 <0.0006 0.0017 <0.0006 <0.0006 0.0032 0.0020 0.0007 <0.0006 A19-61.1 0.0709 0.0128 0.0111 0.0237 0.0160 0.0223 0.0116 0.0123 0.0111 0.0105 0.0111 0.0108 0.0101 A19-46.1 0.0398 0.0203 0.0149 0.0257 0.0277 0.0275 0.0195 0.0271 0.0115 0.0950 0.0618 0.0571 0.1014 g/mL) LY-COV555 0.0071 0.0034 0.0038 0.0093 0.0129 0.0080 0.0032 0.0125 0.0041 >10 >10 >10 >10

50 ( CB6 0.0262 0.0310 0.0172 0.0079 0.0363 0.0432 0.0163 0.0828 0.0401 0.0619 >10 >10 >10 REGN10989 0.0043 0.0033 0.0027 0.0067 0.0036 <0.0006 0.0021 0.0013 0.0001 >10 >10 >10 >10 IC REGN10933 0.0077 0.0052 0.0057 0.0058 >10 0.0074 0.0043 0.0067 0.0069 0.2022 >10 >10 >10 REGN10987 0.0710 0.0200 0.0197 2.0054 0.0291 0.0440 0.0078 0.0237 0.0168 0.0175 0.0264 0.0244 0.0143

B.1.351 B.1.351 WA-1 D614G Var2 Var3 Var4 Var5 Var6 Var7 B.1.1.7 Var9 Var10 Var11 v2 v1 A23-58.1 0.0107 0.0049 0.0039 0.0062 0.0078 0.0049 0.0053 0.0048 0.0039 0.0251 0.0183 0.0091 0.0148 B1-182.1 0.0088 0.0026 0.0021 0.0029 0.0036 <0.0006 0.0032 0.0038 0.0024 0.0099 0.0046 0.0026 0.0038 A19-61.1 0.1633 0.0261 0.0196 0.0418 0.0332 0.0343 0.0202 0.0258 0.0186 0.0255 0.0242 0.0196 0.0170 A19-46.1 0.1287 0.0435 0.0476 0.0636 0.0742 0.0628 0.0603 0.0528 0.0250 0.0255 0.0242 0.1572 0.2069 g/mL) LY-COV555 0.0357 0.0105 0.0145 0.0299 0.0495 0.0222 0.0121 0.0317 0.0159 >10 >10 >10 >10 CB6 0.1139 0.0831 0.0631 0.0282 0.1602 0.3721 0.0987 0.2500 1.5123 0.2342 >10 >10 >10 80 ( REGN10989 0.0087 0.0058 0.0060 0.0110 0.0062 0.0033 0.0032 0.0026 0.0008 >10 >10 >10 >10 IC REGN10933 0.0211 0.0157 0.0129 0.0196 >10 0.0204 0.0150 0.0243 0.0233 0.7863 >10 >10 >10 REGN10987 0.8555 0.4122 0.1870 >10 0.2023 0.3399 0.0674 0.0980 0.2321 0.3040 0.3702 0.1015 0.1621

D K417N E484K N501Y N501Y S982A A570D D80A L18F Δ144 R246I

D614G D614G Δ69-70 Δ242-244 D215G

D1118H A701V

T716I B.1.1.7 B.1.351

Fig. 2. Neutralization and binding activity against spike proteins from circulating variants.

(A) Table showing domain and mutations relative to WA-1 for each of the 13 variants tested in panels B-C.

(B) Spike protein variants were expressed on the surface of HEK293T cells and binding to the indicated antibody was measured using flow cytometry. Data is shown as Mean Fluorescence intensity (MFI) normalized to the MFI for the same antibody against the D614G parental variant. Percent change is indicated by a color gradient from red (increased binding, Max 250%) to white (no change, 100%) to blue (no binding, 0%). Black indicates binding was not tested.

(C) IC50 and IC80 values for the indicated antibodies against WA-1 and the 12 variants shown in (A). Ranges are indicated by colors white (>10 μg/mL), light blue (1-10 μg/mL), yellow (0.1-1 μg/mL), orange (0.05-0.1 μg/mL), red (0.01-0.05 μg/mL), maroon (0.001-0.01 μg/mL) and purple (<0.001 μg/mL). Black indicates a variant that was not tested.

(D) Location of spike protein variant mutations on the spike glycoprotein for B.1.1.7 (left) and B.1.351 (right). P681H is not resolved in the structure and therefore its location is not noted in B.1.1.7. bioRxiv preprint doi: https://doi.org/10.1101/2021.02.25.432969; this version posted March 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license.

Figure 3

A Cryo-EM structure of spike in complex with A23-58.1 B Interactions between A23-58.1 and RBD

Fab A23-58.1 CDR L2 CDR L1 CDR L3 W96 Heavy Y91 Light Epitope of W50 Y32 chain chain A23-58.1 N56 CDR H3 E484 F486 CDR H1 E484 CDR H2 F100F Y489 D100D K417 T478 CDR H2 CDR L1 β6 N487 CDR H1 CDR L2 P95 CDR H3 CDR L3 Y453 β5 S477 N501 F456 Y473 N439

RBD RBD Membrane CDR L1 CDR L2 CDR L3 CDR H1 CDR H2 CDR H3 C Paratope of A23-58.1 Heavy chain ------FR1------_CDR1------FR2----______CDR2______------FR3------______CDR3______-FR4 1 22 31 36 5052A 66 82ABC 92 100ABCDEF 103 | | | | | || | |||| | ||||||| | A23-58.1 QMQLVQSGPEVKKPGTSVKVSCKASGFTFTSSAVQWVRQARGQRLEWIGWIVVGSGNTNYAQKFQERVTITRDMSTSTAYMELSSLRSEDTAVYYCAAPNCSNVVCYDGFDIWGQGTMVTVSS B1-182.1 QMQLVQSGPEVKKPGTSVKVSCKASGFTFTSSAVQWVRQARGQRLEWIGWIVVGSGNTNYAQKFQERVTITRDMSTSTAYMELSSLRSEDTAVYYCAAPYCSGGSCFDGFDIWGQGTMVTVSS S2-E12 QVQLVQSGPEVKKPGTSVRVSCKASGFTFTSSAVQWVRQARGQRLEWVGWIVVGSGNTNYAQKFHERVTITRDMSTSTAYMELSSLRSEDTAVYYCASPYCSGGSCSDGFDIWGQGTMVTVSS Light chain ------FR1------_____CDR1_____------FR2-----__CDR2_------FR3------___CDR3___----FR4--— 1 23 27A 35 50 56 88 98 107 | | || | | | | | | A23-58.1 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYSASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYFCQQYGTSPWTFGQGTKVEIK B1-182.1 EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGFPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQY GNSPWTFGQGTKVEIR S2-E12 DIVLTQTPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYVGLTGWTFGQGTKVEIK D Comparison of epitopes of A23-58.1, S2E12, LY-CoV555, REGN10933 and CB6 Residue # 401 417 439 453 473 484 501 | | | | | | | RBD sequence VIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYR A23-58.1 * ** * ****** ******* * S2E12 * **** * ****** ********* REGN19033 * * * * * ** * **** ******* *** * * * CB6 * ** * *** ** * * ****** ***** ** ** *** * ** ** LY-CoV555 * * ** * * ****** ** *** E Different binding modes of A23-58.1 and REGN10933 F Different binding modes of A23-58.1 and LY-CoV555 A23-58.1 A23-58.1 LY-CoV555 RBD tip REGN-10933 Epitope of Epitope of E484 REGN-10933 LY-CoV555 E484

K417 K417 RBD Y453 saddle Y453

N501 N501

N439 N439

Membrane RBD Membrane RBD

Fig. 3. Structural basis of A23-58.1 binding and neutralization.

(A) Cryo-EM structure of A23-58.1 Fab in complex with SARS-CoV-2 HexaPro spike. Overall density map is shown to the left with protomers colored light green, gray and cyan. One of the A23-58.1 Fab bound to the RBD is shown in orange and blue. Structure of the RBD and A23-58.1 after local focused refinement was shown to the right. The heavy chain CDRs are colored brown, salmon and orange for CDR H1, CDR H2 and CDR H3, respectively. The light chain CDRs are colored marine blue, light blue and purple blue for CDR L1, CDR L2 and CDR L3, respectively. The contour level of Cryo-EM map is 5.7s. (B) Interaction between A23-58.1 and RBD. All CDRs were involved in binding of RBD. Epitope of A23-58.1 is shown in bright green surface with a yellow border (left, viewing from antibody to RBD). RBD mutations in current circulating SARS-CoV-2 variants are colored red. Lys417 and Glu484 are located at the edge of the epitope. The tip of the RBD binds to a cavity formed by the CDRs (right, viewing down to the cavity). Interactions between aromatic/hydrophobic residues are prominent at the lower part of the cavity. Hydrogen bonds at the rim of the cavity are marked with dashed lines. RBD residues were labeled with italicized font. (C) Paratope of A23-58.1. Sequences of B1-182.1 and S2E12 were aligned with variant residues underlined. Paratope residues for A23-58.1 and S2E12 were highlighted in green and light brown, respectively. (D) Epitope of A23-58.1 on RBD. Epitope residues for different RDB-targeting antibodies are marked with * under the RBD sequence. (E) Comparison of binding modes of A23-58.1 and REGN10933. One Fab is shown to bind to the RBD on the spike. The shift of the binding site to the saddle of RBD encircled Lys417, Glu484 and Tyr453 inside the REGN10933 epitope (violet), explaining its sensitivity to the K417N, Y453F and E484K mutations. (F) Comparison of binding modes of A23-58.1 and LY-CoV555. One Fab is shown to bind to the RBD on the spike. Glu484 is located in the middle of LY-CoV555 epitope (light orange), explaining its sensitivity to the E484K mutation. bioRxiv preprint doi: https://doi.org/10.1101/2021.02.25.432969; this version posted March 1, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license.

Figure 4

A Cell Surface Binding to Selected Mutational Sites A23-58.1 MFI Normalized B1-182.1 to WA-1 A19-61.1 250% 200% A19-46.1 150% LY-COV555 100% CB6 50%

REGN10989 0% REGN10933

WA-1F456RA475R T478IF486RN487RY489RQ493RL452RF490LS494RD614G

B D614G/S477N Impact of mutations on antibody neutralization D614G/ D614/ WA-1 F456R A475R T478I F486R N487R L452R F490L S494R K417N S477N A23-58.1 0.0035 0.0220 0.0086 0.0139 >10 >10 0.0032 0.0081 0.0042 0.0007 0.0028 B1-182.1 0.0015 0.0072 0.0080 0.0081 >10 >10 0.0016 0.0027 0.0020 <0.0006 0.0033 A19-61.1 0.0356 0.0184 0.0140 0.0636 0.0776 0.0095 0.0220 0.0267 >10 0.0085 0.0381

g/mL) A19-46.1 0.0213 0.0438 0.0143 0.0601 0.0279 0.0115 >10 >10 >10 0.0127 0.0407

μ LY-COV555 0.0121 0.0298 0.0176 0.0188 1.1810 0.0063 >10 >10 >10 0.0026 0.0079 CB6 0.0280 >10 >10 0.0213 0.4682 >10 0.0175 0.1174 0.0864 >10 0.0180 50 ( REGN10989 0.0022 0.0052 0.0015 0.0013 0.5077 0.0012 0.0031 >10 0.0030 0.0016 0.0036 IC REGN10933 0.0061 0.0231 0.3415 0.0078 >10 >10 0.0058 0.0269 0.0060 0.0540 0.0168 REGN10987 0.0427 0.0239 0.0231 0.0609 0.0188 0.0035 0.2154 0.0577 0.2270 0.0056 0.0294

D614G/ D614/ WA-1 F456RA475R T478IF486RN487RL452RF490L S494R K417N S477N A23-58.1 0.0089 0.0583 0.0296 0.0509 >10 >10 0.0070 0.0153 0.0171 0.0035 0.0072 B1-182.1 0.0078 0.0256 0.0261 0.0420 >10 >10 0.0061 0.0049 0.0069 0.0019 0.0070 A19-61.1 0.1152 0.1041 0.0945 0.1905 0.2087 0.0335 0.0358 0.0527 >10 0.0175 0.0422

g/mL) A19-46.1 0.0821 0.0781 0.0714 0.2092 0.1928 0.0258 >10 >10 >10 0.0361 0.0898

μ LY-COV555 0.0296 0.1530 0.0384 0.0497 >10 0.0154 >10 >10 >10 0.0064 0.0192 CB6 0.1187 >10 >10 0.0974 2.2585 >10 0.1074 0.2337 0.3358 >10 0.0733 80 ( REGN10989 0.0067 0.0102 0.0031 0.0064 4.2766 0.0037 0.0102 >10 0.0071 0.0030 0.0075

IC REGN10933 0.0394 0.0554 1.3074 0.0266 >10 >10 0.0223 0.0609 0.0295 0.5349 0.0454 REGN10987 0.1609 0.0438 0.1296 0.5800 0.1602 0.0249 1.1719 0.6939 2.3085 0.0197 0.1929

C rVSV-SARS CoV2 S mutants E Rate of in vitro resistance acquisition A19-46.1 A19-61.1 A23-58.1 B1-182.1 1 50 A19-46.1 A19-61.1 0.75 5

0.5 0.5 0.25 Frequency 0.05 B1-182.1/A19-61.1 g/mL ]

μ B1-182.1/A19-46.1 0 S S [ V R 0.005 Y449 N450 F490V K444E G446 G593 F486 0.0005 D B1-182.1 Combinations S Max Conc.with >20% CP E 0.00005 B1-182.1 1 2 3 4 5 Selection Round A19-46.1 A19-61.1

Spike

Fig. 4. Critical binding residue determination and mitigation of escape risk using dual antibody combinations

(A) The indicated Spike protein mutations predicted by structural analysis were expressed on the surface of HEK293T cells and binding to the indicated antibody was measured using flow cytometry. Data is shown as Mean Fluorescence Intensity (MFI) normalized to the MFI for the same antibody against the WA-1 parental binding. Percent change is indicated by a color gradient from red (increased binding, Max 250%) to white (no change, 100%) to blue (no binding, 0%).

(B) IC50 and IC80 values for the indicated antibodies against WA-1 and the 10 mutations. Ranges are indicated by colors white (>10 μg/mL), light blue (1-10 μg/mL), yellow (0.1-1 μg/mL), orange (0.05-0.1 μg/mL), red (0.01-0.05 μg/mL), maroon (0.001-0.01 μg/mL) and purple (<0.001 μg/mL).

(C) Replication competent vesicular stomatitis virus (rcVSV) whose genome expressed SARS-CoV-2 WA-1 was incubated with serial dilutions of the indicated antibodies and wells with cytopathic effect (CPE) were passaged forward into subsequent rounds (Figure S6) after 48-72 hours. Total supernatant RNA was harvested and viral genomes shotgun sequenced to determine the frequency of amino acid changes. Shown are the spike protein amino acid/position change and frequency as a logo plot.

(D) Negative stain 3D reconstruction of the ternary complex of spike with Fab B1-182.1 and A19-46.1 (left) or A19-61.1 (right).

(E) rcVSV SARS-CoV-2 was incubated with increasing concentrations (1.3e-4 to 50 μg/mL) of either single antibodies (A19-46.1, A19-61.1 and B1-182.1) and combinations of antibodies (B1-182.1/A19-46.1 and B1-182.1/A19-61.1). Every 3 days, wells were assessed for CPE and the highest concentration well with the >20% CPE was passaged forward onto fresh cells and antibody containing media. Shown is the maximum concentration with >20% CPE for each of the test conditions in each round of selection. Once 50 μg/mL has been reached, virus was no longer passaged forward and a dashed line is used to indicate maximum antibody concentration was reached in subsequent rounds.